This proposal focuses on creation of a sequencing core for the Undiagnosed Disease Program (UDP) as well as comparison of the utility of genome-wide sequencing (GWS; also known as whole genome sequencing) versus Whole Exome Sequencing (WES) for the identification of causal variants. Illumina and the Medical College of Wisconsin (MCW) have worked together to advance genomic sequencing into clinical medicine; this proposal is joint between these entities. All of the necessary components for the UDP sequencing core are functional at MCW and Illumina and required capacity and turnaround are met. Both groups have championed GWS as opposed to WES for genetic discovery leading to the second focus; comparison of GWS and WES for diagnostic success. MCW uses both WES and GWS; along with obvious advantages in detecting non protein coding variants, we find significantly better coverage of actionable genes with GWS, and a higher diagnostic success rate. We thus propose to conduct GWS for all participants enrolled in the UDP creating the opportunity to compare utility of WES versus GWS. With an integrated team and using innovative lab and bioinformatics techniques we propose to test the hypothesis that GWS will produce at least 25% more diagnoses than WES. Aim 1 will generate clinical grade GWS for all UDP cases sequenced and perform read mapping and variant calling. Sharing of the data generated and the methods developed will enable the UDP network to directly compare diagnostic use of WES and GWS. Aim 2 will undertake clinical grade tertiary analysis of the data using our clinically validated analysis platform; we will also provide clinical interpretation and report generation for all cases requested. These will be produced using our existing clinical methodology and tools. Aim 2 will also support dissemination of the methodology and offer tertiary analysis and clinical interpretation to all UDN sites. Aim 3 will confirm the NextGen sequencing results using Sanger and, through gathering of this data, determine whether this step will be necessary in the future. We envision that all of the laboratory operations, methodologies, and tools developed will be made available and will be suited for cloning in additional currently non network hospitals and large clinics. Relevance: This application is highly relevant in that it seeks to establish MCW as the sequencing core for the UDP. In addition to meeting this goal, the application seeks to extend the UDN benefit by determining whether application of GWS as compared to WES provides a diagnostic advantage.